Primate embryonic stem cell line

ABSTRACT

A purified preparation of primate embryonic stem cells is disclosed. This preparation is characterized by the following cell surface markers: SSEA-1 (−); SSEA-4 (+); TRA-1-60 (+); TRA-1-81 (+); and alkaline phosphatase (+). In a particularly advantageous embodiment, the cells of the preparation are human embryonic stem cells, have normal karyotypes, and continue to proliferate in an undifferentiated state after continuous culture for eleven months. The embryonic stem cell lines also retain the ability, throughout the culture, to form trophoblast and to differentiate into all tissues derived from all three embryonic germ layers (endoderm, mesoderm and ectoderm). A method for isolating a primate embryonic stem cell line is also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/982,637, filedOct. 18, 2001 now U.S. Pat. No. 7,029,913, which was a continuation ofU.S. Ser. No. 09/761,289 filed Jan. 16, 2001, now abandoned, which was acontinuation of U.S. Ser. No. 09/106,390 filed June 26, 1998, now U.S.Pat. No. 6,200,806, which was a continuation of U.S. Ser. No. 08/591,246filed Jan. 18, 1996, now U.S. Pat. No. 5,843,780, which was acontinuation-in-part of U.S. Ser. No. 08/376,327 filed Jan. 20, 1995,now abandoned.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States government support awarded byNIH NCRR Grant No. RR00167. The United States government has certainrights in this invention.

BACKGROUND OF THE INVENTION

In general, the field of the present invention is stem cell cultures.Specifically, the field of the present invention is primate embryonicstem cell cultures.

In general, stem cells are undifferentiated cells which can give rise toa succession of mature functional cells. For example, a hematopoieticstem cell may give rise to any of the different types of terminallydifferentiated blood cells. Embryonic stem (ES) cells are derived fromthe embryo and are pluripotent, thus possessing the capability ofdeveloping into any organ or tissue type or, at least potentially, intoa complete embryo.

One of the seminal achievements of mammalian embryology of the lastdecade is the routine insertion of specific genes into the mouse genomethrough the use of mouse ES cells. This alteration has created a bridgebetween the in vitro manipulations of molecular biology and anunderstanding of gene function in the intact animal. Mouse ES cells areundifferentiated, pluripotent cells derived in vitro frompreimplantation embryos (Evans, et al. Nature 292: 154-159, 1981;Martin, Proc. Natl. Acad. Sci. USA 78: 7634-7638, 1981) or from fetalgerm cells (Matsui, et al., Cell 70: 841-847, 1992). Mouse ES cellsmaintain an undifferentiated state through serial passages when culturedin the presence of fibroblast feeder layers in the presence of LeukemiaInhibitory Factor (LIF) (Williams, et al., Nature 336: 684-687, 1988).If LIF is removed, mouse ES cells differentiate.

Mouse ES cells cultured in non-attaching conditions aggregate anddifferentiate into simple embryoid bodies, with an outer layer ofendoderm and an inner core of primitive ectoderm. If these embryoidbodies are then allowed to attach onto a tissue culture surface,disorganized differentiation occurs of various cell types, includingnerves, blood cells, muscle, and cartilage (Martin, 1981, supra;Doetschman, et al., J. Embryol. Exp. Morph. 87: 27-45, 1985). Mouse EScells injected into syngeneic mice form teratocarcinomas that exhibitdisorganized differentiation, often with representatives of all threeembryonic germ layers. Mouse ES cells combined into chimeras with normalpreimplantation embryos and returned to the uterus participate in normaldevelopment (Richard, et al., Cytogenet. Cell Genet. 65: 169-171, 1994).

The ability of mouse ES cells to contribute to functional germ cells inchimeras provides a method for introducing site-specific mutations intomouse lines. With appropriate transfection and selection strategies,homologous recombination can be used to derive ES cell lines withplanned alterations of specific genes. These genetically altered cellscan be used to form chimeras with normal embryos and chimeric animalsare recovered. If the ES cells contribute to the germ line in thechimeric animal, then in the next generation a mouse line for theplanned mutation is established.

Because mouse ES cells have the potential to differentiate into any celltype in the body, mouse ES cells allow the in vitro study of themechanisms controlling the differentiation of specific cells or tissues.Although the study of mouse ES cells provides clues to understanding thedifferentiation of general mammalian tissues, dramatic differences inprimate and mouse development of specific lineages limits the usefulnessof mouse ES cells as a model of human development. Mouse and primateembryos differ meaningfully in the timing of expression of the embryonicgenome, in the formation of an egg cylinder versus an embryonic disc(Kaufman, The Atlas of Mouse Development, London: Academic Press, 1992),in the proposed derivation of some early lineages (O'Rahilly & Muller,Developmental Stages in Human Embryos, Washington: Carnegie Institutionof Washington, 1987), and in the structure and function in theextraembryonic membranes and placenta (Mossman, Vertebrate FetalMembranes, New Brunswick: Rutgers, 1987). Other tissues differ in growthfactor requirements for development (e.g. the hematopoietic system(Lapidot et al., Lab An Sci 43: 147-149, 1994)), and in adult structureand function (e.g. the central nervous system). Because humans areprimates, and development is remarkably similar among primates, primateES cells lines will provide a faithful model for understanding thedifferentiation of primate tissues in general and human tissues inparticular.

The placenta provides just one example of how primate ES cells willprovide an accurate model of human development that cannot be providedby ES cells from other species. The placenta and extraembryonicmembranes differ dramatically between mice and humans. Structurally, themouse placenta is classified as labyrinthine, whereas the human and therhesus monkey placenta are classified as villous. Chorionicgonadotropin, expressed by the trophoblast, is an essential moleculeinvolved in maternal recognition of pregnancy in all primates, includinghumans (Heam, J Reprod Fertil 76: 809-819, 1986; Heam et al., J ReprodFert 92: 497-509, 1991). Trophoblast secretion of chorionic gonadotropinin primates maintains the corpus luteum of pregnancy and, thus,progesterone secretion. Without progesterone, pregnancy fails. Yet mousetrophoblast produces no chorionic gonadotropin, and mice use entirelydifferent mechanisms for pregnancy maintenance (Heam et al., “Normal andabnormal embryo-fetal development in mammals,” In: Lamming E, ed.Marshall's Physiology of Reproduction. 4th ed. Edinburgh, N.Y.:Churchill Livingstone, 535-676, 1994). An immortal, euploid, primate EScell line with the developmental potential to form trophoblast in vitro,will allow the study of the ontogeny and function of genes such aschorionic gonadotropin which are critically important in humanpregnancy. Indeed, the differentiation of any tissue for which there aresignificant differences between mice and primates will be moreaccurately reflected in vitro by primate ES cells than by mouse EScells.

The major in vitro models for studying trophoblast function includehuman choriocarcinoma cells, which are malignant cells that may notfaithfully reflect normal trophectoderm; short-term primary cultures ofhuman and non-human primate cytotrophoblast, which in present cultureconditions quickly form non-dividing syncytial trophoblast; and in vitroculture of preimplantation non-human primate embryos (Hearn, et al., J.Endocrinol. 119: 249-255, 1988; Coutifaris, et al., Ann. NY Acad. Sci.191-201, 1994). An immortal, euploid, non-human primate embryonic stem(ES) cell line with the developmental potential to form trophectodermoffers significant advantages over present in vitro models of humantrophectoderm development and function, as trophoblast-specific genessuch as chorionic gonadotropin could be stably altered in the ES cellsand then studied during differentiation to trophectoderm.

The cell lines currently available that resembles primate ES cells mostclosely are human embryonic carcinoma (EC) cells, which are pluripotent,immortal cells derived from teratocarcinomas (Andrews, et al., Lab.Invest. 50 (2): 147-162, 1984; Andrews, et al., in: Robertson E., ed.Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. Oxford:IRL press, pp. 207-246, 1987). EC cells can be induced to differentiatein culture, and the differentiation is characterized by the loss ofspecific cell surface markers (SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81)and the appearance of new markers (Andrews, et al., 1987, supra). HumanEC cells will form teratocarcinomas with derivatives of multipleembryonic lineages in tumors in nude mice. However, the range ofdifferentiation of these human EC cells is limited compared to the rangeof differentiation obtained with mouse ES cells, and all EC cell linesderived to date are aneuploid (Andrews, et al., 1987, supra). Similarmouse EC cell lines have been derived from teratocarcinomas, and, ingeneral their developmental potential is much more limited than mouse EScells (Rossant, et al., Cell Differ. 15: 155-161, 1984).Teratocarcinomas are tumors derived from germ cells, and although germcells (like ES cells) are theoretically totipotent (i.e. capable offorming all cell types in the body), the more limited developmentalpotential and the abnormal karyotypes of EC cells are thought to resultfrom selective pressures in the teratocarcinoma tumor environment(Rossant & Papaioannou, Cell Differ 15: 155-161, 1984). ES cells, on theother hand, are thought to retain greater developmental potentialbecause they are derived from normal embryonic cells in vitro, withoutthe selective pressures of the teratocarcinoma environment. Nonetheless,mouse EC cells and mouse ES cells share the same unique combination ofcell surface markers (SSEA-1 (+), SSEA-3 (−), SSEA-4 (−), and alkalinephosphatase (+)).

Pluripotent cell lines have also been derived from preimplantationembryos of several domestic and laboratory animals species (Evans, etal., Theriogenology 33 (1): 125-128, 1990; Evans, et al., Theriogenology33 (1): 125-128, 1990; Notarianni, et al., J. Reprod. Fertil. 41(Suppl.): 51-56, 1990; Giles, et al., Mol. Reprod. Dev. 36: 130-138,1993; Graves, et al., Mol. Reprod. Dev. 36: 424-433, 1993; Sukoyan, etal., Mol. Reprod. Dev. 33: 418-431, 1992; Sukoyan, et al., Mol. Reprod.Dev. 36: 148-158, 1993; Iannaccone, et al., Dev. Biol. 163: 288-292,1994).

Whether or not these cell lines are true ES cells lines is a subjectabout which there may be some difference of opinion. True ES cellsshould: (i) be capable of indefinite proliferation in vitro in anundifferentiated state; (ii) maintain a normal karyotype throughprolonged culture; and (iii) maintain the potential to differentiate toderivatives of all three embryonic germ layers (endoderm, mesoderm, andectoderm) even after prolonged culture. Strong evidence of theserequired properties have been published only for rodents ES cellsincluding mouse (Evans & Kaufman, Nature 292: 154-156, 1981; Martin,Proc Natl Acad Sci USA 78: 7634-7638, 1981) hamster (Doetschman et al.Dev Biol 127: 224-227, 1988), and rat (lannaccone et al. Dev Biol 163:288-292, 1994), and less conclusively for rabbit ES cells (Giles et al.Mol Reprod Dev 36: 130-138, 1993; Graves & Moreadith, Mol Reprod Dev 36:424-433, 1993). However, only established ES cell lines from the rat(Iannaccone, et al., 1994, supra) and the mouse (Bradley, et al, Nature309: 255-256, 1984) have been reported to participate in normaldevelopment in chimeras. There are no reports of the derivation of anyprimate ES cell line.

BRIEF SUMMARY OF THE INVENTION

The present invention is a purified preparation of primate embryonicstem cells. The primate ES cell lines are true ES cell lines in thatthey: (i) are capable of indefinite proliferation in vitro in anundifferentiated state; (ii) are capable of differentiation toderivatives of all three embryonic germ layers (endoderm, mesoderm, andectoderm) even after prolonged culture; and (iii) maintain a normalkaryotype throughout prolonged culture. The true primate ES cells linesare therefore pluripotent.

The present invention is also summarized in that primate ES cell linesare preferably negative for the SSEA-1 marker, preferably positive forthe SSEA-3 marker, and positive for the SSEA-4 marker. The primate EScell lines are also positive for the TRA-1-60, and TRA-1-81 markers, aswell as positive for the alkaline phosphatase marker.

It is an advantageous feature of the present invention that the primateES cell lines continue to proliferate in an undifferentiated state aftercontinuous culture for at least one year. In a particularly advantageousembodiment, the cells remain euploid after proliferation in anundifferentiated state.

It is a feature of the primate ES cell lines in accordance with thepresent invention that the cells can differentiate to trophoblast invitro and express chorionic gonadotropin.

The present invention is also a purified preparation of primateembryonic stem cells that has the ability to differentiate into cellsderived from mesoderm, endoderm, and ectoderm germ layers after thecells have been injected into an immunocompromised mouse, such as a SCIDmouse.

The present invention is also a method of isolating a primate embryonicstem cell line. The method comprises the steps of isolating a primateblastocyst, isolating cells from the inner cellular mass (ICM) of theblastocyst, plating the ICM cells on a fibroblast layer (whereinICM-derived cell masses are formed) removing an ICM-derived cell massand dissociating the mass into dissociated cells, replating thedissociated cells on embryonic feeder cells and selecting colonies withcompact morphology containing cells with a high nucleus/cytoplasm ratio,and prominent nucleoli. The cells of the selected colonies are thencultured.

It is an object of the present invention to provide a primate embryonicstem cell line.

It is an object of the present invention to provide a primate embryonicstem cell line characterized by the following markers: alkalinephosphatase(+); SSEA-1(−); preferably SSEA-3(+); SSEA-4(+); TRA-1-60(+);and TRA-1-81(+).

It is an object of the present invention to provide a primate embryonicstem cell line capable of proliferation in an undifferentiated stateafter continuous culture for at least one year. Preferably, these cellsremain euploid.

It is another object of the present invention to provide a primateembryonic stem cell line wherein the cells differentiate into cellsderived from mesoderm, endoderm, and ectoderm germ layers when the cellsare injected into an immunocompromised mouse.

Other objects, features, and advantages of the present invention willbecome obvious after study of the specification, drawings, and claims.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a photomicrograph illustrating normal XY karyotype of rhesusES cell line R278.5 after 11 months of continuous culture.

FIGS. 2A-2D are a set of phase-contrast photomicrographs demonstratingthe morphology of undifferentiated rhesus ES (R278.5) cells and of cellsdifferentiated from R278.5 in vitro (bar=100μ). Photograph 2Ademonstrates the distinct cell borders, high nucleus to cytoplasm ratio,and prominent nucleoli of undifferentiated rhesus ES cells. Photographs2B-2D shows differentiated cells eight days after plating R278.5 cellson gel treated tissue culture plastic (with 10³ units/ml added humanLIF). Cells of these three distinct morphologies are consistentlypresent when R278.5 cells are allowed to differentiate at low densitywithout fibroblasts either in the presence or absence of soluble humanLIF.

FIGS. 3A-F are photomicrographs demonstrating the expression of cellsurface markers on undifferentiated rhesus ES (R278.5) cells (bar=100μ).Photograph 3A shows Alkaline Phosphatase (+); Photograph 3B shows SSEA-1(−); Photograph 3C shows SSEA-3 (+); Photograph 3D shows SSEA-4 (+);Photograph 3E shows TRA-1-60 (+); and Photograph 3F shows TRA-1-81 (+).

FIGS. 4A-4B are photographs illustrating expression of α-fetoproteinmRNA and α- and β-chorionic gonadotrophin mRNA expression in rhesus EScells (R278.5) allowed to differentiate in culture.

FIGS. 5A-5F include six photomicrographs of sections of tumors formed byinjection of 0.5×10⁶ rhesus ES (R278.5) cells into the hindleg musclesof SCID mice and analyzed 15 weeks later. Photograph 5A shows a lowpower field demonstrating disorganized differentiation of multiple celltypes. A gut-like structure is encircled by smooth muscle(s), andelsewhere foci of cartilage (c) are present (bar=400μ); Photograph 5Bshows striated muscle (bar=40μ); Photograph 5C shows stratified squamousepithelium with several hair follicles. The labeled hair follicle (f)has a visible hair shaft (bar=200μ); Photograph 5D shows stratifiedlayers of neural cells in the pattern of a developing neural tube. Anupper “ventricular” layer, containing numerous mitotic figures (arrows),overlies a lower “mantle” layer. (bar=100μ); Photograph 5E showsciliated columnar epithelium (bar=40μ); Photograph 5F shows villicovered with columnar epithelium with interspersed mucus-secretinggoblet cells (bar=200μ).

FIGS. 6A-6B include photographs of an embryoid Body. This embryoid bodywas formed from a marmoset ES cell line (Cj62) that had beencontinuously passaged in vitro for over 6 months. Photograph 6A (above)shows a section of the anterior ⅓ of the embryonic disc. Note theprimitive ectoderm (E) forms a distinct cell layer from the underlyingprimitive endoderm (e), with no mixing of the cell layers. Note alsothat amnion (a) is composed of two distinct layers; the inner layer iscontinuous with the primitive ectoderm at the margins. Photograph 6B(below) shows a section in the caudal ⅓ of embryonic disc. Note centralgroove (arrow) and mixing of primitive ectoderm and endodermrepresenting early primitive streak formation, indicating the beginningof gastrulation. 400×, toluidine blue stain.

DETAILED DESCRIPTION OF THE INVENTION

(1) In General

(a) Uses of Primate ES Cells

The present invention is a pluripotent, immortal euploid primate ES cellline, as exemplified by the isolation of ES cell lines from two primatespecies, the common marmoset (Callithrix jacchus) and the rhesus monkey(Macaca mulatta). Primate embryonic stem cells are useful for:

-   -   (i) Generating transgenic non-human primates for models of        specific human genetic diseases. Primate embryonic stem cells        will allow the generation of primate tissue or animal models for        any human genetic disease for which the responsible gene has        been cloned. The human genome project will identify an        increasing number of genes related to human disease, but will        not always provide insights into gene function. Transgenic        nonhuman primates will be essential for elucidating mechanisms        of disease and for testing new therapies.    -   (ii) Tissue transplantation. By manipulating culture conditions,        primate ES cells, human and non-human, can be induced to        differentiate to specific cell types, such as blood cells,        neuron cells, or muscle cells. Alternatively, primate ES cells        can be allowed to differentiate in tumors in SCID mice, the        tumors can be disassociated, and the specific differentiated        cell types of interest can be selected by the usage of lineage        specific markers through the use of fluorescent activated cell        sorting (FACS) or other sorting method or by direct        microdissection of tissues of interest. These differentiated        cells could then be transplanted back to the adult animal to        treat specific diseases, such as hematopoietic disorders,        endocrine deficiencies, degenerative neurological disorders or        hair loss.

(b) Selection of Model Species

Macaques and marmosets were used as exemplary species for isolation of aprimate ES cell line. Macaques, such as the rhesus monkey, are Old Worldspecies that are the major primates used in biomedical research. Theyare relatively large (about 7-10 kg). Males take 4-5 years to mature,and females have single young. Because of the extremely close anatomicaland physiological similarities between humans and rhesus monkeys, rhesusmonkey true ES cell lines provide a very accurate in vitro model forhuman differentiation. Rhesus monkey ES cell lines and rhesus monkeyswill be particularly useful in the testing of the safety and efficacy ofthe transplantation of differentiated cell types into whole animals forthe treatment of specific diseases or conditions. In addition, thetechniques developed for the rhesus ES cell lines model the generation,characterization and manipulation of human ES cell lines.

The common marmoset (Callithrix jacchus) is a New World primate specieswith reproductive characteristics that make it an excellent choice forES cell derivation. Marmosets are small (about 350-400 g), have a shortgestation period (144 days), reach sexual maturity in about 18 months,and routinely have twins or triplets. Unlike in macaques, it is possibleto routinely synchronize ovarian cycles in the marmoset withprostaglandin analogs, making collection of age-matched embryos frommultiple females possible, and allowing efficient embryo transfer tosynchronized recipients with 70%-80% of embryos transferred resulting inpregnancies. Because of these reproductive characteristics that allowfor the routine efficient transfer of multiple embryos, marmosetsprovide an excellent primate species in which to generate transgenicmodels for human diseases.

There are approximately 200 primate species in the world. The mostfundamental division that divides higher primates is between Old Worldand New world species. The evolutionary distance between the rhesusmonkey and the common marmoset is far greater than the evolutionarydistance between humans and rhesus monkeys. Because it is heredemonstrated that it is possible to isolate ES cell lines from arepresentative species of both the Old World and New World group usingsimilar conditions, the techniques described below may be usedsuccessfully in deriving ES cell lines in other higher primates as well.Given the close evolutionary distance between rhesus macaques andhumans, and the fact that feeder-dependent human EC cell lines can begrown in conditions similar to those that support primate ES cell lines,the same growth conditions will allow the isolation and growth of humanES cells. In addition, human ES cell lines will be permanent cell linesthat will also be distinguished from all other permanent human celllines by their normal karyotype and the expression of the samecombination of cell surface markers (alkaline phosphotase, preferablySSEA-3, SSEA-4, TRA-1-60 and TRA-1-81) that characterize other primateES cell lines. A normal karyotype and the expression of this combinationof cell surface markers will be defining properties of true human EScell lines, regardless of the method used for their isolation andregardless of their tissue of origin.

No other primate (human or non-human) ES cell line is known to exist.The only published permanent, euploid, embryo-derived cell lines thathave been convincingly demonstrated to differentiate into derivatives ofall three germ layers have been derived from rodents (the mouse, rat,and hamster), and possibly from rabbit. The published reports ofembryo-derived cell lines from domestic species have failed toconvincingly demonstrate differentiation of derivatives of all threeembryonic germ layers or have not been permanent cell lines. Researchgroups in Britain and Singapore are informally reported, later than thework described here, to have attempted to derive human ES cell linesfrom surplus in vitro fertilization-produced human embryos, althoughthey have not yet reported success in demonstrating pluripotency oftheir cells and have failed to isolate permanent cell lines. In the onlypublished report on attempts to isolate human ES cells, conditions wereused (LIF in the absence of fibroblast feeder layers) that the resultsbelow will indicate will not result in primate ES cells which can remainin an undifferentiated state. It is not surprising, then that the cellsgrown out of human ICMs failed to continue to proliferate after 1 or 2subcultures, Bongso et al. Hum. Reprod. 9: 2100-2117 (1994).

(2) Embryonic Stem Cell Isolation

A preferable medium for isolation of embryonic stem cells is “ESmedium.” ES medium consists of 80% Dulbecco's modified Eagle's medium(DMEM; no pyruvate, high glucose formulation, Gibco BRL), with 20% fetalbovine serum (FBS; Hyclone), 0.1 mM β-mercaptoethanol (Sigma), 1%non-essential amino acid stock (Gibco BRL). Preferably, fetal bovineserum batches are compared by testing clonal plating efficiency of a lowpassage mouse ES cell line (ES_(jt3)), a cell line developed just forthe purpose of this test. FBS batches must be compared because it hasbeen found that batches vary dramatically in their ability to supportembryonic cell growth, but any other method of assaying the competenceof FBS batches for support of embryonic cells will work as analternative.

Primate ES cells are isolated on a confluent layer of murine embryonicfibroblast in the presence of ES cell medium. Embryonic fibroblasts arepreferably obtained from 12 day old fetuses from outbred CF1 mice(SASCO), but other strains may be used as an alternative. Tissue culturedishes are preferably treated with 0.1% gelatin (type I; Sigma).

For rhesus monkey embryos, adult female rhesus monkeys (greater thanfour years old) demonstrating normal ovarian cycles are observed dailyfor evidence of menstrual bleeding (day 1 of cycle=the day of onset ofmenses). Blood samples are drawn daily during the follicular phasestarting from day 8 of the menstrual cycle, and serum concentrations ofluteinizing hormone are determined by radioimmunoassay. The female ispaired with a male rhesus monkey of proven fertility from day 9 of themenstrual cycle until 48 hours after the luteinizing hormone surge;ovulation is taken as the day following the luteinizing hormone surge.Expanded blastocysts are collected by non-surgical uterine flushing atsix days after ovulation. This procedure routinely results in therecovery of an average 0.4 to 0.6 viable embryos per rhesus monkey permonth, Seshagiri et al. Am J Primatol 29: 81-91, 1993.

For marmoset embryos, adult female marmosets (greater than two years ofage) demonstrating regular ovarian cycles are maintained in familygroups, with a fertile male and up to five progeny. Ovarian cycles arecontrolled by intramuscular injection of 0.75 g of the prostaglandinPGF2a analog cloprostenol (Estrumate, Mobay Corp, Shawnee, Kans.) duringthe middle to late luteal phase. Blood samples are drawn on day 0(immediately before cloprostenol injection), and on days 3, 7, 9, 11,and 13. Plasma progesterone concentrations are determined by ELISA. Theday of ovulation is taken as the day preceding a plasma progesteroneconcentration of 10 ng/ml or more. At eight days after ovulation,expanded blastocysts are recovered by a non-surgical uterine flushprocedure, Thomson et al. “Non-surgical uterine stage preimplantationembryo collection from the common marmoset,” J Med Primatol, 23: 333-336(1994). This procedure results in the average production of 1.0 viableembryos per marmoset per month.

The zona pellucida is removed from blastocysts by brief exposure topronase (Sigma). For immunosurgery, blastocysts are exposed to a 1:50dilution of rabbit anti-marmoset spleen cell antiserum (for marmosetblastocysts) or a 1:50 dilution of rabbit anti-rhesus monkey (for rhesusmonkey blastocysts) in DMEM for 30 minutes, then washed for 5 minutesthree times in DMEM, then exposed to a 1:5 dilution of Guinea pigcomplement (Gibco) for 3 minutes.

After two further washes in DMEM, lysed trophectoderm cells are removedfrom the intact inner cell mass (ICM) by gentle pipetting, and the ICMplated on mouse inactivated (3000 rads gamma irradiation) embryonicfibroblasts.

After 7-21 days, ICM-derived masses are removed from endoderm outgrowthswith a micropipette with direct observation under a stereo microscope,exposed to 0.05% Trypsin-EDTA (Gibco) supplemented with 1% chicken serumfor 3-5 minutes and gently dissociated by gentle pipetting through aflame polished micropipette.

Dissociated cells are replated on embryonic feeder layers in fresh ESmedium, and observed for colony formation. Colonies demonstratingES-like morphology are individually selected, and split again asdescribed above. The ES-like morphology is defined as compact colonieshaving a high nucleus to cytoplasm ratio and prominent nucleoli.Resulting ES cells are then routinely split by brief trypsinization orexposure to Dulbecco's Phosphate Buffered Saline (without calcium ormagnesium and with 2 mM EDTA) every 1-2 weeks as the cultures becomedense. Early passage cells are also frozen and stored in liquidnitrogen.

Cell lines may be karyotyped with a standard G-banding technique (suchas by the Cytogenetics Laboratory of the University of Wisconsin StateHygiene Laboratory, which provides routine karyotyping services) andcompared to published karyotypes for the primate species.

Isolation of ES cell lines from other primate species would follow asimilar procedure, except that the rate of development to blastocyst canvary by a few days between species, and the rate of development of thecultured ICMs will vary between species. For example, six days afterovulation, rhesus monkey embryos are at the expanded blastocyst stage,whereas marmoset embryos don't reach the same stage until 7-8 days afterovulation. The Rhesus ES cell lines were obtained by splitting theICM-derived cells for the first time at 7-16 days after immunosurgery;whereas the marmoset ES cells were derived with the initial split at7-10 days after immunosurgery. Because other primates also vary in theirdevelopmental rate, the timing of embryo collection, and the timing ofthe initial ICM split will vary between primate species, but the sametechniques and culture conditions will allow ES cell isolation.

Because ethical considerations in the U.S. do not allow the recovery ofhuman in vivo fertilized preimplantation embryos from the uterus, humanES cells that are derived from preimplantation embryos will be derivedfrom in vitro fertilized (IVF) embryos. Experiments on unused (spare)human IVF-produced embryos are allowed in many countries, such asSingapore and the United Kingdom, if the embryos are less than 14 daysold. Only high quality embryos are suitable for ES isolation. Presentdefined culture conditions for culturing the one cell human embryo tothe expanded blastocyst are suboptimal but practicable, Bongso et al.,Hum Reprod 4: 706-713, 1989. Co-culturing of human embryos with humanoviductal cells results in the production of high blastocyst quality.IVF-derived expanded human blastocysts grown in cellular co-culture, orin improved defined medium, will allow the isolation of human ES cellswith the same procedures described above for nonhuman primates.

(3) Defining Characteristics of Primate ES Cells

Primate embryonic stem cells share features with the primate ICM andwith pluripotent human embryonal carcinoma cells. Putative primate EScells may therefore be characterized by morphology and by the expressionof cell surface markers characteristic of human EC cells. Additionally,putative primate ES cells may be characterized by developmentalpotential, karyotype and immortality.

(a) Morphology

The colony morphology of primate embryonic stem cell lines is similarto, but distinct from, mouse embryonic stem cells. Both mouse andprimate ES cells have the characteristic features of undifferentiatedstem cells, with high nuclear/cytoplasmic ratios, prominent nucleoli,and compact colony formation. The colonies of primate ES cells areflatter than mouse ES cell colonies and individual primate ES cells canbe easily distinguished. In FIG. 2, reference character A indicates aphase contrast photomicrograph of cell line R278.5 demonstrating thecharacteristic primate ES cell morphology.

(b) Cell Surface Markers

A primate ES cell line of the present invention is distinct from mouseES cell lines by the presence or absence of the cell surface markersdescribed below.

One set of glycolipid cell surface markers is known as theStage-specific embryonic antigens 1 through 4. These antigens can beidentified using antibodies for SSEA 1, preferably SSEA-3 and SSEA-4which are available from the Developmental Studies Hybridoma Bank of theNational Institute of Child Health and Human Development. The cellsurface markers referred to as TRA-1-60 and TRA-1-81 designateantibodies from hybridomas developed by Peter Andrews of the Universityof Sheffield and are described in Andrews et al., “Cell lines from humangerm cell tumors,” In: Robertson E, ed. Teratocarcinomas and EmbryonicStem Cells: A Practical Approach. Oxford: IRL Press, 207-246, 1987. Theantibodies were localized with a biotinylated secondary antibody andthen an avidin/biotinylated horseradish peroxidase complex (VectastainABC System, Vector Laboratories). Alternatively, it should also beunderstood that other antibodies for these same cell surface markers canbe generated. NTERA-2 cl. D1, a pluripotent human EC cell line (gift ofPeter Andrews), may be used as a negative control for SSEA-1, and as apositive control for SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. This cellline was chosen for positive control only because it has beenextensively studied and reported in the literature, but other human ECcell lines may be used as well.

Mouse ES cells (ES_(jt3)) are used as a positive control for SSEA-1, andfor a negative control for SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Otherroutine negative controls include omission of the primary or secondaryantibody and substitution of a primary antibody with an unrelatedspecificity.

Alkaline phosphatase may be detected following fixation of cells with 4%para-formaldehyde using “Vector Red” (Vector Laboratories) as asubstrate, as described by the manufacturer (Vector Laboratories). Theprecipitate formed by this substrate is red when viewed with a rhodaminefilter system, providing substantial amplification over lightmicroscopy.

Table 1 diagrams a comparison of mouse ES cells, primate ES cells, andhuman EC cells. The only cells reported to express the combination ofmarkers SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 other than primate EScells are human EC cells. The globo-series glycolipids SSEA-3 and SSEA-4are consistently present on human EC cells, and are of diagnostic valuein distinguishing human EC cell tumors from human yolk sac carcinomas,choriocarcinomas, and other lineages which lack these markers, Wenk etal., Int J Cancer 58:108-115, 1994. A recent survey found SSEA-3 andSSEA-4 to be present on all of over 40 human EC cell lines examined,Wenk et al. TRA-1-60 and TRA-1-81 antigens have been studied extensivelyon a particular pluripotent human EC cell line, NTERA-2 CL. D1, Andrewset al, supra. Differentiation of NTERA-2 CL. D1 cells in vitro resultsin the loss of SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 expression and theincreased expression of the lacto-series glycolipid SSEA-1, Andrews etal, supra. This contrasts with undifferentiated mouse ES cells, whichexpress SSEA-1, and neither SSEA-3 nor SSEA-4. Although the function ofthese antigens are unknown, their shared expression by R278.5 cells andhuman EC cells suggests a close embryological similarity. Alkalinephosphatase will also be present on all primate ES cells. A successfulprimate ES cell culture of the present invention will correlate with thecell surface markers found in the rhesus macaque and marmoset cell linesdescribed in Table 1.

As disclosed below in Table 1, the rhesus macaque and marmoset celllines are identical to human EC cell lines for the 5 described markers.Therefore, a successful primate ES cell culture will also mimic human ECcells. However, there are other ways to discriminate ES cells from ECcells. For example, the primate ES cell line has a normal karyotype andthe human EC cell line is aneuploid.

In FIG. 3, the photographs labelled A through F demonstrate thecharacteristic staining of these markers on a rhesus monkey ES cell linedesignated R278.5.

TABLE 1 Mouse C. jacchus M. mulatta Human EC ES ES ES (NTERA-2 cl.D1)SSEA-1 + − − − SSEA-3 − + + + SSEA-4 − + + + Tra-1-60 − + + + Tra-1-81− + + +

(c) Developmental Potential

Primate ES cells of the present invention are pluripotent. By“pluripotent” we mean that the cell has the ability to develop into anycell derived from the three main germ cell layers or an embryo itself.When injected into SCID mice, a successful primate ES cell line willdifferentiate into cells derived from all three embryonic germ layersincluding: bone, cartilage, smooth muscle, striated muscle, andhematopoietic cells (mesoderm); liver, primitive gut and respiratoryepithelium (endoderm); neurons, glial cells, hair follicles, and toothbuds (ectoderm).

This experiment can be accomplished by injecting approximately0.5-1.0×10⁶ primate ES cells into the rear leg muscles of 8-12 week oldmale SCID mice. The resulting tumors can be fixed in 4% paraformaldehydeand examined histologically after paraffin embedding at 8-16 weeks ofdevelopment. In FIG. 4, photomicrographs designated A-F are of sectionsof tumors formed by injection of rhesus ES cells into the hind legmuscles of SCID mice and analyzed 15 weeks later demonstratingcartilage, smooth muscle, and striated muscle (mesoderm); stratifiedsquamous epithelium with hair follicles, neural tube with ventricular,intermediate, and mantle layers (ectoderm); ciliated columnar epitheliumand villi lined by absorptive enterocytes and mucus-secreting gobletcells (endoderm).

A successful nonhuman primate ES cell line will have the ability toparticipate in normal development when combined in chimeras with normalpreimplantation embryos. Chimeras between preimplantation nonhumanprimate embryos and nonhuman primate ES cells can be formed by routinemethods in several ways. (i) injection chimeras: 10-15 nonhuman primateES cells can be microinjected into the cavity of an expanded nonhumanprimate blastocyst; (ii) aggregation chimeras: nonhuman primate morulaecan be co-cultured on a lawn of nonhuman primate ES cells and allowed toaggregate; and (iii) tetraploid chimeras: 10-15 nonhuman primate EScells can be aggregated with tetraploid nonhuman primate morulaeobtained by electrofusion of 2-cell embryos, or incubation of morulae inthe cytoskeletal inhibitor cholchicine. The chimeras can be returned tothe uterus of a female nonhuman primate and allowed to develop to term,and the ES cells will contribute to normal differentiated tissuesderived from all three embryonic germ layers and to germ cells. Becausenonhuman primate ES can be genetically manipulated prior to chimeraformation by standard techniques, chimera formation followed by embryotransfer can lead to the production of transgenic nonhuman primates.

(d) Karyotype

Successful primate ES cell lines have normal karyotypes. Both XX and XYcells lines will be derived. The normal karyotypes in primate ES celllines will be in contrast to the abnormal karyotype found in humanembryonal carcinoma (EC), which are derived from spontaneously arisinghuman germ cell tumors (teratocarcinomas). Human embryonal carcinomacells have a limited ability to differentiate into multiple cell typesand represent the closest existing cell lines to primate ES cells.Although tumor-derived human embryonal carcinoma cell lines have someproperties in common with embryonic stem cell lines, all human embryonalcarcinoma cell lines derived to date are aneuploid. Thus, primate EScell lines and human EC cell lines can be distinguished by the normalkaryotypes found in primate ES cell lines and the abnormal karyotypesfound in human EC lines. By “normal karyotype” it is meant that allchromosomes normally characteristic of the species are present and havenot been noticeably altered.

Because of the abnormal karyotypes of human embryonal carcinoma cells,it is not clear how accurately their differentiation reflects normaldifferentiation. The range of embryonic and extra-embryonicdifferentiation observed with primate ES cells will typically exceedthat observed in any human embryonal carcinoma cell line, and the normalkaryotypes of the primate ES cells suggests that this differentiationaccurately recapitulates normal differentiation.

(e) Immortality

Immortal cells are capable of continuous indefinite replication invitro. Continued proliferation for longer than one year of culture is asufficient evidence for immortality, as primary cell cultures withoutthis property fail to continuously divide for this length of time(Freshney, Culture of animal cells. New York: Wiley-Liss, 1994). PrimateES cells will continue to proliferate in vitro with the cultureconditions described above for longer than one year, and will maintainthe developmental potential to contribute all three embryonic germlayers. This developmental potential can be demonstrated by theinjection of ES cells that have been cultured for a prolonged period(over a year) into SCID mice and then histologically examining theresulting tumors. Although karyotypic changes can occur randomly withprolonged culture, some primate ES cells will maintain a normalkaryotype for longer than a year of continuous culture.

(f) Culture Conditions

Growth factor requirements to prevent differentiation are different forthe primate ES cell line of the present invention than the requirementsfor mouse ES cell lines. In the absence of fibroblast feeder layers,Leukemia inhibitory factor (LIF) is necessary and sufficient to preventdifferentiation of mouse ES cells and to allow their continuous passage.Large concentrations of cloned LIF fail to prevent differentiation ofprimate ES cell lines in the absence of fibroblast feeder layers. Inthis regard, primate ES stem cells are again more similar to human ECcells than to mouse ES cells, as the growth of feeder-dependent human ECcells lines is not supported by LIF in the absence of fibroblasts.

(g) Differentiation to Extra Embryonic Tissues

When grown on embryonic fibroblasts and allowed to grow for two weeksafter achieving confluence (i.e., continuously covering the culturesurface), primate ES cells of the present invention spontaneouslydifferentiate and will produce chorionic gonadotropin, indicatingtrophoblast differentiation (a component of the placenta) and produceα-fetoprotein, indicating endoderm differentiation. Chorionicgonadotropin activity can be assayed in the medium conditioned bydifferentiated cells by Leydig cell bioassay, Seshagiri & Hearn, HumReprod 8: 279-287, 1992. For mRNA analysis, RNA can be prepared byguanidine isothiocyanate-phenol/chloroform extraction (1) fromapproximately 0.2×10⁶ differentiated cells and from 0.2×10⁶undifferentiated cells. The relative levels of the mRNA forα-fetoprotein and the α- and β-subunit of chorionic gonadotropinrelative to glyceraldehyde-3-phosphate dehydrogenase can be determinedby semi-quantitative Reverse Transcriptase-Polymerase Chain Reaction(RT-PCR). The PCR primers for glyceraldehyde 3-phosphate dehydrogenase(G3PDH), obtained from Clontech (Palo Alto, Calif.), are based on thehuman cDNA sequence, and do not amplify mouse G3PDH mRNA under ourconditions. Primers for the α-fetoprotein mRNA are based on the humansequence and flank the 7th intron (5′ primer=(5′)GCTGGATTGTCTGCAGGATGGGGAA (SEQ ID NO: 1); 3′ primer=(5′)TCCCCTGAAGAAAATTGGTTAAAAT (SEQ ID NO: 2)). They amplify a cDNA of 216nucleotides. Primers for the β-subunit of chorionic gonadotropin flankthe second intron (5′ primer=(5′) ggatc CACCGTCAACACCACCATCTGTGC (SEQ IDNO: 3); 3′ primer=(5′) ggatc CACAGGTCAAAGGGTGGTCCTTGGG (SEQ ID NO: 4))(nucleotides added to the hCGb sequence to facilitate sub-cloning areshown in lower case italics). They amplify a cDNA of 262 base pairs. Theprimers for the CGα subunit can be based on sequences of the first andfourth exon of the rhesus gene (5′ primer=(5′) gggaattcGCAGTTACTGAGAACTCACAAG (SEQ ID NO: 5); 3′ primer=(5′) gggaattcGAAGCATGTCAAAGTGGTATGG (SEQ ID NO: 6)) and amplify a cDNA of 556 basepairs. The identity of the α-fetoprotein, CGα and CGβ cDNAs can beverified by subcloning and sequencing.

For Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR), 1 to 5 μlof total R278.5 RNA can be reverse transcribed as described Golos et al.Endocrinology 133 (4): 1744-1752, 1993, and one to 20 μl of reversetranscription reaction was then subjected to the polymerase chainreaction in a mixture containing 1-12.5 pmol of each G3PDH primer, 10-25pmol of each mRNA specific primer, 0.25 mM dNTPs (Pharmacia, Piscataway,N.J.), 1×AmpliTaq buffer (final reaction concentrations=10 mM Tris, pH8.3, 50 mM KCl, 1.5 mM MgCl2, 0.001% (w/v) gelatin) 2.5 μCi ofdeoxycytidine 5′a[³²P]triphosphate (DuPont, Boston, Mass.), 10% glyceroland 1.25 U of AmpliTaq (Perkin-Elmer, Oak Brook, Ill.) in a total volumeof 50 μl. The number of amplification rounds which produced linearincreases in target cDNAs and the relation between input RNA and amountof PCR product is empirically determined as by Golos et al. Samples werefractionated in 3% Nusieve (FMC, Rockland, Me.) agarose gels (1×TBErunning buffer) and DNA bands of interest were cut out, melted at 65° C.in 0.5 ml TE, and radioactivity determined by liquid scintillationcounting. The ratio of counts per minute in a specific PCR productrelative to cpm of G3PDH PCR product is used to estimate the relativelevels of a mRNAs among differentiated and undifferentiated cells.

The ability to differentiate into trophectoderm in vitro and the abilityof these differentiated cells to produce chorionic gonadotropindistinguishes the primate ES cell line of the present invention from allother published ES cell lines.

EXAMPLES

(1) Animals and Embryos

As described above, we have developed a technique for non-surgical,uterine-stage embryo recovery from the rhesus macaque and the commonmarmoset.

To supply rhesus embryos to interested investigators, The WisconsinRegional Primate Research Center (WRPRC) provides a preimplantationembryo recovery service for the rhesus monkey, using the non-surgicalflush procedure described above. During 1994, 151 uterine flushes wereattempted from rhesus monkeys, yielding 80 viable embryos (0.53 embryosper flush attempt).

By synchronizing the reproductive cycles of several marmosets,significant numbers of in vivo produced, age-matched, preimplantationprimate embryos were studied in controlled experiments for the firsttime. Using marmosets from the self-sustaining colony (250 animals) ofthe Wisconsin Regional Primate Research Center (WRPRC), we recovered 54viable morulae or blastocysts, 7 unfertilized oocytes or degenerateembryos, and 5 empty zonae pellucidae in a total of 54 flush attempts(1.0 viable embryo-flush attempt). Marmosets have a 28 day ovariancycle, and because this is a non-surgical procedure, females can beflushed on consecutive months, dramatically increasing the embryo yieldcompared to surgical techniques which require months of rest betweencollections.

(2) Rhesus Macaque Embryonic Stem Cells

Using the techniques described above, we have derived three independentembryonic stem cell lines from two rhesus monkey blastocysts (R278.5,R366, and R367). One of these, R278.5, remains undifferentiated andcontinues to proliferate after continuous culture for over one year.R278.5 cells have also been frozen and successfully thawed with therecovery of viable cells.

The morphology and cell surface markers of R278.5 cells areindistinguishable from human EC cells, and differ significantly frommouse ES cells. R278.5 cells have a high nucleus/cytoplasm ratio andprominent nucleoli, but rather than forming compact, piled-up colonieswith indistinct cell borders similar to mouse ES cells, R278.5 cellsform flatter colonies with individual, distinct cells (FIG. 2A). R278.5cells express the SSEA-3, SSEA-4, TRA-1-60, and TRA-81 antigens (FIG. 3and Table 1), none of which are expressed by mouse ES cells. The onlycells known to express the combination of markers SSEA-3, SSEA-4,TRA-1-60, and TRA-1-81 other than primate ES cells are human EC cells.The globo-series glycolipids SSEA-3 and SSEA-4 are consistently presenton human EC cells, and are of diagnostic value in distinguishing humanEC cell tumors from yolk sac carcinomas, choriocarcinomas and other stemcells derived from human germ cell tumors which lack these markers, Wenket al, Int J Cancer 58: 108-115, 1994. A recent survey found SSEA-3 andSSEA-4 to be present on all of over 40 human EC cell lines examined(Wenk et al.).

TRA-1-60 and TRA-1-81 antigens have been studied extensively on aparticular pluripotent human EC cell line, NTERA-2 CL. D1 (Andrews etal.). Differentiation of NTERA-2 CL. D1 cells in vitro results in theloss of SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81 expression and theincreased expression of the lacto-series glycolipid SSEA-1.Undifferentiated mouse ES cells, on the other hand, express SSEA-1, andnot SSEA-3, SSEA-4, TRA-1-60 or TRA-1-81 (Wenk et al.). Although thefunction of these antigens is unknown, their expression by R278.5 cellssuggests a close embryological similarity between primate ES cells andhuman EC cells, and fundamental differences between primate ES cells andmouse ES cells.

R278.5 cells also express alkaline phosphatase. The expression ofalkaline phosphatase is shared by both primate and mouse ES cells, andrelatively few embryonic cells express this enzyme. Positive cellsinclude the ICM and primitive ectoderm (which are the most similarembryonic cells in the intact embryo to ES cells), germ cells, (whichare totipotent), and a very limited number of neural precursors, KaufmanM H. The atlas of mouse development. London: Academic Press, 1992. Cellsnot expressing this enzyme will not be primate ES cells.

Although cloned human LIF was present in the medium at cell linederivation and for initial passages, R278.5 cells grown on mouseembryonic fibroblasts without exogenous LIF remain undifferentiated andcontinued to proliferate. R278.5 cells plated on gelatin-treated tissueculture plates without fibroblasts differentiated to multiple cell typesor failed to attach and died, regardless of the presence or absence ofexogenously added human LIF (FIG. 2). Up to 10⁴ units/ml human LIF failsto prevent differentiation. In addition, added LIF fails to increase thecloning efficiency or proliferation rate of R278.5 cells on fibroblasts.Since the derivation of the R278.5 cell line, we have derived twoadditional rhesus ES cell lines (R366 and R367) on embryonic fibroblastswithout any exogenously added LIF at initial derivation. R366 and R367cells, like R278.5 cells, continue to proliferate on embryonicfibroblasts without exogenously added LIF and differentiate in theabsence of fibroblasts, regardless of the presence of added LIF. RT-PCRperformed on mRNA from spontaneously differentiated R278.5 cellsrevealed α-fetoprotein mRNA (FIG. 4). α-fetoprotein is a specific markerfor endoderm, and is expressed by both extra-embryonic (yolk sac) andembryonic (fetal liver and intestines) endoderm-derived tissues.Epithelial cells resembling extraembryonic endoderm are present in cellsdifferentiated in vitro from R278.5 cells (FIG. 2). Bioactive CG (3.89ml units/ml) was present in culture medium collected from differentiatedcells, but not in medium collected from undifferentiated cells (lessthan 0.03 ml units/ml), indicating the differentiation of trophoblast, atrophectoderm derivative. The relative level of the CGα mRNA increased23.9-fold after differentiation (FIG. 4).

All SCID mice injected with R278.5 cells in either intra-muscular orintra-testicular sites formed tumors, and tumors in both sitesdemonstrated a similar range of differentiation. The oldest tumorsexamined (15 weeks) had the most advanced differentiation, and all hadabundant, unambiguous derivatives of all three embryonic germ layers,including gut and respiratory epithelium (endoderm); bone, cartilage,smooth muscle, striated muscle (mesoderm); ganglia, glia, neuralprecursors, and stratified squamous epithelium (ectoderm), and otherunidentified cell types (FIG. 5). In addition to individual cell types,there was organized development of some structures which require complexinteractions between different cell types. Such structures included gutlined by villi with both absorptive enterocytes and mucus-secretinggoblet cells, and sometimes encircled by layers of smooth muscle in thesame orientation as muscularis mucosae (circular) and muscularis (outerlongitudinal layer and inner circular layer); neural tubes withventricular, intermediate, and mantle layers; and hair follicles withhair shafts (FIG. 5).

The essential characteristics that define R278.5 cells as ES cellsinclude: indefinite (greater than one year) undifferentiatedproliferation in vitro, normal karyotype, and potential to differentiateto derivatives of trophectoderm and all three embryonic germ layers. Inthe mouse embryo, the last cells capable of contributing to derivativesof both trophectoderm and ICM are early ICM cells. The timing ofcommitment to ICM or trophectoderm has not been established for anyprimate species, but the potential of rhesus ES cells to contribute toderivatives of both suggests that they most closely resemble earlytotipotent embryonic cells. The ability of rhesus ES cells to formtrophoblast in vitro distinguishes primate ES cell lines from mouse EScells. Mouse ES cell have not been demonstrated to form trophoblast invitro, and mouse trophoblast does not produce gonadotropin. Rhesus EScells and mouse ES cells do demonstrate the similar wide range ofdifferentiation in tumors that distinguishes ES cells from EC cells. Thedevelopment of structures composed of multiple cell types such as hairfollicles, which require inductive interactions between the embryonicepidermis and underlying mesenchyme, demonstrates the ability of rhesusES cells to participate in complex developmental processes.

The rhesus ES lines R366 and R367 have also been further cultured andanalyzed. Both lines have a normal XY karyotype and were proliferated inan undifferentiated state for about three months prior to freezing forlater analysis. Samples of each of the cell lines R366 and R367 wereinjected into SCID mice which then formed teratomas identical to thoseformed by R278.5 cells. An additional rhesus cell line R394 having anormal XX karyotype was also recovered. All three of these cell lines,R366, R367 and R394 are identical in morphology, growth characteristics,culture requirements and in vitro differentiation characteristics, i.e.the trait of differentiation to multiple cell types in the absence offibroblasts, to cell line 278.5.

It has been determined that LIF is not required either to derive orproliferate these ES cultures. Each of the cell lines R366, R367 andR394 were derived and cultured without exogenous LIF.

It has also been demonstrated that the particular source of fibroblastsfor co-culture is not critical. Several fibroblast cell lines have beentested both with rhesus line R278.5 and with the marmoset cell linesdescribed below. The fibroblasts tested include mouse STO cells (ATCC56-X), mouse 3T3 cells (ATCC 48-X), primary rhesus monkey embryonicfibroblasts derived from 36 day rhesus fetuses, and mouse Sl/Sl⁴ cells,which are deficient in the steel factor. All these fibroblast cell lineswere capable of maintaining the stem cell lines in an undifferentiatedstate. Most rapid proliferation of the stem cells was observed usingprimary mouse embryonic fibroblasts.

Unlike mouse ES cells, neither rhesus ES cells nor feeder-dependenthuman EC cells remain undifferentiated and proliferate in the presenceof soluble human LIF without fibroblasts. The factors that fibroblastsproduce that prevent the differentiation of rhesus ES cells orfeeder-dependent human EC cells are unknown, but the lack of adependence on LIF is another characteristic that distinguishes primateES cells from mouse ES cells. The growth of rhesus monkey ES cells inculture conditions similar to those required by feeder-dependent humanEC cells, and the identical morphology and cell surface markers ofrhesus ES cells and human EC cells, suggests that similar cultureconditions will support human ES cells.

Rhesus ES cells will be important for elucidating the mechanisms thatcontrol the differentiation of specific primate cell types. Given theclose evolutionary distance and the developmental and physiologicalsimilarities between humans and rhesus monkeys, the mechanismscontrolling the differentiation of rhesus cells will be very similar tothe mechanisms controlling the differentiation of human cells. Theimportance of elucidating these mechanisms is that once they areunderstood, it will be possible to direct primate ES cells todifferentiate to specific cell types in vitro, and these specific celltypes can be used for transplantation to treat specific diseases.

Because ES cells have the developmental potential to give rise to anydifferentiated cell type, any disease that results in part or in wholefrom the failure (either genetic or acquired) of specific cell typeswill be potentially treatable through the transplantation of cellsderived from ES cells. Rhesus ES cells and rhesus monkeys will beinvaluable for testing the efficacy and safety of the transplantation ofspecific cell types derived from ES cells. A few examples of humandiseases potentially treatable by this approach with human ES cellsinclude degenerative neurological disorders such as Parkinson's disease(dopanergic neurons), juvenile onset diabetes (pancreatic β-islet cells)or Acquired Immunodeficiency Disease (lymphocytes). Becauseundifferentiated ES cells can proliferate indefinitely in vitro, theycan be genetically manipulated with standard techniques either toprevent immune rejection after transplantation, or to give them newgenetic properties to combat specific diseases. For specific cell typeswhere immune rejection can be prevented, cells derived from rhesusmonkey ES cells or other non-human primate ES cells could be used fortransplantation to humans to treat specific diseases.

(3) Marmoset Embryonic Stem Cells

Our method for creating an embryonic stem cell line is described above.Using isolated ICM's derived by immunosurgery from marmoset blastocysts,we have isolated 7 putative ES cell lines, each of which have beencultured for over 6 months.

One of these, Cj11, was cultured continuously for over 14 months, andthen frozen for later analysis. The Cj11 cell line and other marmoset EScell lines have been successfully frozen and then thawed with therecovery of viable cells. These cells have a high nuclear/cytoplasmicratio, prominent nucleoli, and a compact colony morphology similar tothe pluripotent human embryonal carcinoma (EC) cell line NT2/D2.

Four of the cell lines we have isolated have normal XX karyotypes, andone has a normal XY karyotype (Karyotypes were performed by Dr. CharlesHarris, University of Wisconsin). These cells were positive for a seriesof cell surface markers (alkaline phosphatase, SSEA-3, SSEA-4, TRA-1-60,and TRA-1-81) that in combination are definitive markers forundifferentiated human embryonal carcinoma cells (EC) cells and primateES cells. In particular, these markers distinguish EC cells from theearliest lineages to differentiate in the human preimplantation embryo,trophectoderm (represented by BeWO choriocarcinoma cells) andextraembryonic endoderm (represented by 1411H yolk sac carcinoma cells).

When the putative marmoset ES cells were removed from fibroblastfeeders, they differentiated into cells of several distinctmorphologies. Among the differentiated cells, trophectoderm is indicatedby the secretion of chorionic gonadotropin and the presence of thechorionic gonadotropin β-subunit mRNA. 12.7 mIU/ml luteinizing hormone(LH) activity was measured in the WRPRC core assay lab using a mouseLeydig cell bioassay in medium conditioned 24 hours by putative ES cellsallowed to differentiate for one week. Note that chorionic gonadotrophinhas both LH and FSH activity, and is routinely measured by LH assays.Control medium from undifferentiated ES cells had less than 1 mIU/ml LHactivity.

Chorionic gonadotropin β-subunit mRNA was detected by reversetranscriptase-polymerase chain reaction (RT-PCR). DNA sequencingconfirmed the identity of the chorionic gonadotrophin β-subunit.

Endoderm differentiation (probably extraembryonic endoderm) wasindicated by the presence of α-fetoprotein mRNA, detected by RT-PCR.

When the marmoset ES cells were grown in high densities, over a periodof weeks epithelial cells differentiated and covered the culture dish.The remaining groups of undifferentiated cells rounded up into compactballs and then formed embryoid bodies (as shown in FIG. 6) thatrecapitulated early development with remarkable fidelity. Over 3-4weeks, some of the embryoid bodies formed a bilaterally symmetricpyriform embryonic disc, an amnion, a yolk sac, and a mesoblastoutgrowth attaching the caudal pole of the amnion to the culture dish.

Histological and ultrastructural examination of one of these embryoidbodies (formed from a cell line that had been passaged continuously for6 months) revealed a remarkable resemblance to a stage 6-7post-implantation embryo. The embryonic disc was composed of apolarized, columnar epithelial epiblast (primitive ectoderm) layerseparated from a visceral endoderm (primitive endoderm) layer. Electronmicroscopy of the epiblast revealed apical junctional complexes, apicalmicrovilli, subapical intermediate filaments, and a basement membraneseparating the epiblast from underlying visceral endoderm. All of theseelements are features of the normal embryonic disc. In the caudal thirdof the embryonic disc, there was a midline groove, disruption of thebasement membrane, and mixing of epiblast cells with underlyingendodermal cells (early primitive streak). The amnion was composed of aninner squamous (ectoderm) layer continuous with the epiblast and anouter mesoderm layer. The bilayered yolk sac had occasionalendothelial-lined spaces containing possible hematopoietic precursors.

The morphology, immortality, karyotype, and cell surface markers ofthese marmoset cells identify these marmoset cells as primate ES cellssimilar to the rhesus ES cells. Since the last cells in the mammalianembryo capable of contributing to both trophectoderm derivatives andendoderm derivatives are the totipotent cells of the early ICM, theability of marmoset ES cells to contribute to both trophoblast andendoderm demonstrates their similarities to early totipotent embryoniccells of the intact embryo. The formation of embryoid bodies by marmosetES cells, with remarkable structural similarities to the earlypost-implantation primate embryo, demonstrates the potential of marmosetES cells to participate in complex developmental processes requiring theinteraction of multiple cell types.

Given the reproductive characteristics of the common marmoset describedabove (efficient embryo transfer, multiple young, short generationtime), marmoset ES cells will be particularly useful for the generationof transgenic primates. Although mice have provided invaluable insightsinto gene function and regulation, the anatomical and physiologicaldifferences between humans and mice limit the usefulness of transgenicmouse models of human diseases. Transgenic primates, in addition toproviding insights into the pathogenesis of specific diseases, willprovide accurate animal models to test the efficacy and safety ofspecific treatments.

1. A pluripotent primate embryonic stem cell line that (i) proliferatesin an in vitro culture for over one year, (ii) maintains the potentialto differentiate to derivatives of endoderm, mesoderm, and ectodermtissues, (iii) is inhibited from differentiation when cultured on afibroblast feeder layer, (iv) is negative for the SSEA-1 cell surfacemarker and positive for alkaline phosphatase and the SSEA-3, Tra-1-60,Tra-1-81, and SSEA-4 cell surface markers, and (v) forms teratomas andnot teratocarcinomas when injected into an immuno-compromised mouse. 2.The cell line of claim 1, wherein the cell line spontaneouslydifferentiates to trophoblast and, wherein said trophoblast produceschorionic gonadotropin when cultured beyond confluence.
 3. The cell lineof claim 2, wherein the cell line differentiates to trophoblast thatproduces chorionic gonadotropin when cultured for two weeks beyondconfluence.
 4. The cell line of claim 1, wherein the cells are andremain euploid for more than one year of continuous culture.
 5. The cellline of claim 1, wherein the cells differentiate into cells derived frommesoderm, endoderm and ectoderm germ layers when injected into a SCIDmouse.
 6. The cell line of claim 1, wherein the cells are human.